In this study we have characterized one of the reactive metabolites of CHCl3 by incubating liver microsomes with (14C) CHCl3 in the presence of cysteine. This thiol reagent was found to block the covalent binding to microsomal protein and concomitantly trap a reactive metabolite of CHCl3. The trapped product was identified as 2-oxothiazolidine-4-carboxylic by reverse isotope dilution and gas chromatography-mass spectrometry (GC-MS). When the incubation was repeated in an atmosphere of 18O2, the trapped product contained virtually 100% 18O2 in the 2-oxo position. These results are consistent with CHCl3 being activated by cytochrome P-450 thru an oxidation of the C-H bond to produce C(OH)Cl3 which would spontaneously dehydrochlorinate to yield phosgene, COC12. This reactive product, which is known to be toxic, could either react with microsomal protein or be trapped by cysteine to produce 2-oxothiazolidine-4-carboxylic acid. A similar oxidative dechlorination reaction leading to the formation of a reactive intermediate has previously been reported by this laboratory for the antibiotic, chloramphenicol. BIBLIOGRAPHIC REFERENCES: Nelson, S.D., Mitchell, J.R. and Pohl, L.R.: Application of chemical ionization MS and the twin-ion technique in the analysis of reactive intermediates in drug metabolism: In Frigerio, A. and Bhisalberti, E.L. (Eds.): Mass Spectrometry in Drug Metabolism. New York, Plenum Publishing Corporation, 1977, pp. 237-249.